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BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF)-DEPENDENT ACTIVATION OF CALPAIN: INVOLVEMENT OF ERK PATHWAY AND REGULATION OF AMPA RECEPTOR FUNCTION AND STABILITY
by
Karoline Rostamiani
_______________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETICS, MOLECULAR, AND CELLULAR BIOLOGY)
May 2011
Copyright 2011 Karoline Rostamiani

Ionotropic glutamate receptors mediate most excitatory synaptic transmission in mammalian brain, and play important roles in neuronal development and synaptic plasticity. In particular, long-term potentiation (LTP) and long-term depression (LTD) are generally considered to represent cellular mechanisms involved in certain forms of learning and memory. Calpain is a calcium-dependent neutral protease that plays significant roles in synaptic plasticity, cell motility, as well as in various forms of neurodegeneration. We compared the rates of calpain-mediated truncation of GluR1 and GluR2 subunits with those of GluR1 phosphorylated at serine 831 or serine 845, and GluR2 phosphorylated at serine 880. Rat brain membranes were treated with calpain and calcium and levels of total and phosphorylated GluR1 and GluR2 subunits were analyzed by western blots. Rates of calpain-mediated truncation of phosphorylated GluR1 (either at serine 831 or 845) were much slower than for total GluR1. On the other hand, the rate of calpain-mediated truncation of GluR2-pS880 was much faster than for GluR2.; We further examined the role of BNDF in calpain activation. In neurons, MAPK can be activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate calpain-2 by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells. Calpain activation was monitored by quantitative analysis of spectrin degradation. In both cell types, BDNF and EGF rapidly activated calpain, and this activation was completely blocked by MAPK and calpain-specific inhibitors. Changes in AMPA receptor function and number at postsynaptic sites have been demonstrated to represent a cellular mechanism for both short-term and long-term plasticity.; AMPA receptor trafficking is a highly regulated process involving many proteins and protein/protein interactions. Transmembrane AMPA receptor regulatory proteins (TARPs) are transmembrane auxiliary proteins known to associate with AMPA receptors and to regulate not only their membrane targeting but also the kinetic properties of the receptors. We investigated whether stargazin could be a calpain substrate in various brain regions and under what conditions could stargazin levels be regulated by calpain activation. Treatment of cortical or cerebellar homogenates with purified calpain and calcium resulted in the rapid degradation of stargazin.

BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF)-DEPENDENT ACTIVATION OF CALPAIN: INVOLVEMENT OF ERK PATHWAY AND REGULATION OF AMPA RECEPTOR FUNCTION AND STABILITY
by
Karoline Rostamiani
_______________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETICS, MOLECULAR, AND CELLULAR BIOLOGY)
May 2011
Copyright 2011 Karoline Rostamiani